Novel Benzosuberone Derivatives: Synthesis, Characterization and Antibacterial Activity

The synthesis of novel amide derivatives of benzosuberone 7a-j from commercially available benzosuberone was successfully achieved in six steps. Some of the important reactions that are involved in the synthesis are (i) insertion of methylester (ii) Suzki reaction and (iii) saponification followed by amide bond formation. The newly synthesis benzosuberone derivatives 7a-j were screened for antibacterial activity and the results indicated that in general, benzosuberone derivatives with R = piperazine ring showed good antibacterial activity.

of a set of new benzosuberone derivatives 7a-j, prepared from commercially available benzosuberone in five steps.

Chemistry
The synthesis of benzosuberone derivatives 7a-j is illustrated in scheme 1. Treatment of benzosuberone 1 with cyanoformate in presence of NaHMDS in THF at 0 o C for 2.5 h yielded in the corresponding methylester 2 in 85% yield. Reaction of compound 2 with N-Phenyl-bis (trifluoromethanesulfonamide)(PhNTf 2 ) in presence of sodium bis(trimethylsilyl)amide in THF at -78 o C to room temperature gave the corresponding triflate derivative 3 in 70% yield. Suzuki reaction of triflate 3 with 3-benzoloxy benzene boric acid in presence of Pd 2 (dba) 3 , Na 2 CO 3 :water, in diethoxymethane, at 90 o C for 2 h yielded benzoloxy methyl ester derivative 4 in 86% yield. Saponification of methyl ester 4 in presence of NaOH:water, in dioxane at 110 o C for 20 h resulted in the carboxylic acid 5 in 92% yield. Coupling of carboxylic acid 5 with secondary amines 6a-j in presence of EDC, HOBT, triethyl amine in dichloromethane at room temperature for 16 h resulted in the corresponding amide derivatives 7a-j. The structures of these compounds were established on the basis of 1 H NMR, mass and IR data. The mass spectral data (M+1) of the compounds are in agreement with desired molecular formulae. As a example, the 1 H NMR of compound 7b i.e (E)-5-(3-(benzyloxy)phenyl) annulene-6-carboxamide is described here: The aromatic ring protons (13 H) appeared in the region 7.40 ppm -6.78 ppm, while the characteristic benzoloxy methylene -CH 2 signal appeared at 5.0 ppm as singlet and the -NH proton signal resonated at 5.18 ppm as a broad singlet. The seven membered methylene protons resonated at 2.56ppm, 2.30 ppm and 2.19 ppm. The mass spectrum of compound 7b showed molecular ion peak at m/z 384.3 M+1 peak.

Antibacterial activity
The results of the antibacterial activity of benzosuberone amides 7a-j is tabulated in table 1. Compounds 7g, 7h, 7i and 7j showed good activity, compounds 7a, 7b, 7e and 7f showed moderate activity and the remaining compounds 7c and 7d displayed weak activity, against the bacterial strains mentioned in the table 1.The zone of inhibitions of compounds 7a-j varied between 17-23 mm and was evaluated with respect to the drug Norfloxacin (zone of inhibitions 19-25 mm). Based on the above results, it may be generalized that benzosuberone derivatives with R = piperazine ring exceptional  being morpholine ring showed good antibacterial activity, while the remaining substituent's in the series (R = -NH 2 , -NHMe, Pyrrolidine, Piperidine, -NMe 2 and -N(CH 2 CH 3 ) 2 exhibited moderate to weak antibacterial activity. Based on the above generalization, it may be predicted that a further structural activity relationship may lead to a good antibacterial drug candidate.

EXPERIMENTAL
Standard operating procedures was implemented for the purification of solvents before being utilized for the reactions and work up's. Merck silica gel 60 (230-400 mesh) and Merck pre-coated plates (silica gel 60 F254) was used for the routine column chromatography and visualization of spots (under UV lamp). Mel-temp apparatus was utilized for the determination of melting point (m.p). Agilent ion trap MS was utilized for recording the mass spectra. Perkin Elmer FT-IR spectrometer was used for recording the IR data. Varian NMR-300 MHz instrument was used to record 1 H NMR spectra. Chemical shifts values are measured in terms of ṕ pm (parts per million) with reference to tetramethylsilane (TMS) as internal standard. The following notations viz., singlet-s, doublet-d, double doublet-dd, and multiplet-m was used for the signals that has appeared in the proton NMR spectrum and the coupling constant value was measured in terms of Hz. methyl 6,7,8,9-tetrahydro-5-oxo-5H-benzo [7] annulene-6-carboxylate 2 A pre-mixed solution of sodium bis-(trimethylsilyl) amide (11.43 g, 62.45 mL, 62.45 mmol) in THF was added for 1h, to benzosuberone 1 (5 g, 31.25 mmol) dissolved in dry THF (50 mL), maintaining the temperature of the reaction at 0 o C. Methyl cyanoformate (3.45 g, 40.58 mmol) was slowly added to the above contents and stirred for 1.5 h at the same same temperature. After completion of the reaction, as monitored by T.L.C, the reaction contents were diluted with saturated NH 4 Cl solution followed by EtOAc. The separated organic layer was worked up in a regular procedure that is extracting with ethylacetate and washing with water followed by bine solution and drying on anhydrous sodium sulphate. Concentration of the ethylacetate layer under reduced pressure resulted in the crude compound. Column chromatography of the crude compound was done (silica gel: 60-120 mesh; elluant: 1-2% EtOAc in pet ether) to afford compound 2. Pale yellow liquid; Yield: 5. (Z)-8-(methoxycarbonyl)-6,7-dihydro-5Hbenzo [7]annulen-9-yl trifluoromethanesulfonate 3 Sodium bis(trimethylsilyl)amide (10.57 g, 57.8 mmol) was dissolved in THF and added to compound 2 (6.3 g, 28.89 mmol) that was previously dissolved in dry THF which was cooled at -78°C. The reaction contents was stirred for 1 h and then N-Phenyl-bis(trifluoromethanesulfonamide) (PhNTf2) (15.475 g, 43.34 mmol) was added to the above reaction mixture at -78°C and was allowed to stir for 2 h at -65 to -55 °C. After reaching the room temperature the reaction mixture was stirred for additional 16 h. After completion of the reaction, as monitored by T.L.C, the reaction contents were diluted with saturated NH 4 Cl solution followed by EtOAc. The separated organic layer was worked up in a regular procedure that is extracting with ethylacetate and washing with water followed by bine solution and drying on anhydrous sodium sulphate. Concentration of the ethylacetate layer under reduced pressure resulted in the crude compound. Column chromatography of the crude compound was done (silica gel: 60-120 mesh; elluant: 1-2% EtOAc in pet ether) to afford

Anti-microbial activity assay
Benzosuberone derivatives 7a-j were tested against the bacterial strains viz., i) Escherichia coli (MTCC443), (ii) Pseudomonas aeruginosa (MTCC424), (Gram negative bacteria) and (iii) Staphylococcus aureus (MTCC96) iv) Streptococcus pyogenes (MTCC442), (Gram positive bacteria) using agar diffusion method according to the literature protocol [10][11][12]. Benzosuberone derivatives 7a-j were dissolved in dimethyl sulphoxide at 25 ¼g/mL concentration. Paper discs (6 mm, punched from Whatmann no 1 paper) were ultraviolet sterilized and used for the assays. Discs were soaked in different concentration of the test solution and placed on the inoculated agar media at regular intervals of 6-7 cm, taking care to ensure that excess solution was not left on the discs. The plates were incubated at 37 o C in an inverted fashion. Activity was determined by zones showing complete inhibition (mm). Growth inhibition was calculated with reference to positive control. All the samples were taken in triplicate.

CONCLUSION
The newly synthesized benzosuberone derivatives 7a-j was determined by spectroscopic techniques like 1 H NMR, mass and IR. From the results of the antibacterial screening data, it was evident that compounds 7g, 7h, 7i and 7j showed good activity and the remaining compounds showed moderate to weak activity.